Techniques for vesicle isolation/purification

At EVs & MS lab, we are specialized in the optimization and set-up of methods for the isolation of extracellular vesicles at the laboratory scale. Generally, we employ classical ultracentrifugation, filtration, chromatography-based techniques, and their combinations, but we also engaged in the development of novel methods for EV isolation. For each starting material, we optimize the isolation protocols in regards to buffer composition, co-purifying contaminants, volume, and initial concentration of the vesicles. We have developed and patented methods for the isolation of urinary EVs suitable for downstream proteomics (G. Pocsfalvi, I. Fiume, D.A.A. Raj, G. Capasso (2011) UIBM N° NA2011A000012) and the isolation of EVs from microalgae (A. Bongiovanni, G. Pocsfalvi, M. Manno, N. Touzet, 2020 PCT/EP2020/086622). 

Ultracentrifugation is one of the most frequently used techniques applied for EV isolation. Differential centrifugation involves a series of low-velocity centrifugation and ultracentrifugation steps to concentrate the EVs in a pelleted fraction. The efficiency of EV isolation by ultracentrifugation depends on many factors, such as acceleration, rotor characteristics (k factor, radius of rotation), sedimentation path length, and viscosity of the sample. Density gradient ultracentrifugation is applied to increase the efficiency of the separation of the particles according to their buoyant density. We use both continuous and stepwise density gradients formed by sucrose, sucrose-deuterium oxide, or iodixanol. 

We use filtration methods in combination with differential or gradient ultracentrifugation. Our protocols rely on the use of commercial ultrafiltration devices, tangential flow cartridges, or molecular filters with different cut-offs using vacuum or centrifugal concentrators. 

We use our in-house protocols to pack gravity columns for size exclusion chromatography (SEC) and employ them for the isolation/purification of EVs. FPLC and HPLC techniques are also available at IBBR-CNR for chromatography-based EV purification. 

LC-ESI-MS/MS and proteomics

We apply gel and in-solution digestion-based proteomics workflows and immunoblotting for the analysis of protein biocargo of EVs.
QSTAR® Elite (Applied Biosystems): a hybrid quadrupole-time-of-flight (Q-TOF) tandem mass spectrometer (MS/MS) equipped with a nano-electrospray ion source and coupled to a 2D nano-HPLC system (Ultimate 3000) equipped with an autosampler. 
Other instrumentations used for protein and carbohydrate and fatty acids analysis are: 
  • Novex® NuPAGE® SDS-PAGE gel and Novex® NativePAGE™ gel systems 
  • iBlot® Dry Blot and iBind™ Western Blot systems 
  • Hewlett Packard, Series 1100 HPLC with UV detector 
  • Perkin-Elmer, Series 200 HPLC system with autosampler 
  • Dionex BioLC® system: a high-performance anion-exchange HPAE chromatography with pulsed amperometry detection (PAD) for carbohydrate analysis. 
  • Ultimate 3000 (LC Packings) 2D-nano-HPLC interfaced with QSTAR-Elite mass spectrometer 
  • TLC system for fatty acid analysis 
For further information, please visit us at 

GC-MS/MS

TSQ™ Duo Triple Quadrupole GC-MS/MS (Thermo Fisher Scientific) interfaced to Trace 1300 GC and an automatic liquid sampler is used for EV metabolomics work. As an example, we have analyzed the metabolites expressed in grapefruit juice-derived nano- and micro-vesicles (DOI: 10.3390/cells9122722). 

For further information, please visit us at https://ibbr.cnr.it/ibbr/facilities//gc-ms-ms 

Protein sequencing

EVs &MS lab performs N-terminal protein/peptide sequencing based on the Edman degradation reaction using a Shimadzu PPSQ 33B sequencer. 

For further information, please visit us at https://ibbr.cnr.it/ibbr/facilities//protein-peptide-sequencing 
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At EVs & MS we are developing a novel class of molecular delivery vectors for human health and next-generation organic farming by building on the natural delivery capabilities of nanovesicles

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Address:

EVs & MS laboratory

Institute of Biosciences and BioResources

National Research Council

via P. Castellino, 111

80131, Napoli

ITALY

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